some polyphenols inhibit the formation of pentyl radical and octanoic acid radical

Biochem. J. (2000) 346, 265–273 (Printed in Great Britain) 265

Some polyphenols inhibit the formation of pentyl radical and octanoicacid radical in the reaction mixture of linoleic acid hydroperoxide withferrous ionsHideo IWAHASHI1

Department of Chemistry, Wakayama Medical College, 811-1 Kimiidera, Wakayama 641-0012, Japan

Effects of some polyphenols and their related compounds

(chlorogenic acid, caffeic acid, quinic acid, ferulic acid, gallic

acid, -()-catechin, -(®)-catechin, 4-hydroxy-3-methoxy-

benzoic acid, salicylic acid, -dopa, dopamine, -adrenaline, -

noradrenaline, o-dihydroxybenzene, m-dihydroxybenzene, and

p-dihydroxybenzene) on the formation of 13-hydroperoxide

octadecadienoic (13-HPODE) acid-derived radicals (pentyl rad-

ical and octanoic acid radical) were examined. The ESR spin

trapping showed that chlorogenic acid, caffeic acid, gallic acid,

-()-catechin, -(®)-catechin, -dopa, dopamine, -adren-

aline, -noradrenaline, and o-dihydroxybenzene inhibited

the overall formation of 13-HPODE acid-derived radicals in the

reaction mixture of 13-HPODE with ferrous ions. The ESR peak

heights of α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN)}13-HPODE-derived radical adducts decreased to 46³4%

(chlorogenic acid), 54³2% (caffeic acid), 49³2% (gallic acid),

55³1% [-()-catechin], 60³3% [-(®)-catechin], 42³1%

INTRODUCTION

Linoleic acid hydroperoxide and hydroperoxides of other un-

saturated fatty acids are extremely toxic when injected into mice

[1]. Subcutaneous and intravenous injection of linoleic acid

peroxide resulted in a marked lesion in intima of the aorta [2,3].

Lipid peroxide-derived radicals seem to be related to the toxicity

of the lipid peroxides. Indeed, lipid-derived free radicals, which

form in the reaction of lipid peroxides with transition metals are

known to cause damage to biomembranes, proteins and the

other biomolecules [4–6]. Products analysis and ESR spin-

trapping investigations provided evidence for alkoxyl radical

intermediates from fatty acid hydroperoxides [7–9]. Furthermore,

pentyl radical and 12,13-epoxylinoleic acid radical, which form

through β-scission and intramolecular epoxidation of the fatty

acid alkoxyl radicals, were detected [10–14].

Chlorogenic acid is an ester of caffeic acid (CA) with quinic

acid. It is found naturally in various agricultural products such

as coffee beans, potatoes, apples and tobacco leaves. Measure-

ments showed chlorogenic acid to be present in significant

quantities : 3±4–14 mg}100 g fresh weight in several varieties of

potatoes [15], 12–31 mg}100 ml of juice produced from apples

[16], 89 mg}100 g of fresh mature apples [17], 559–674 mg}100 g

of dry tea shoots [18]. Catechol derivatives such as caffeic acid

are also present at about 250 mg per cup of coffee [19]. Catechin,

epicatechin and epicatechin gallate are abundantly contained in

green tea and grape seeds. Epicatechin was also detected in red

wine and grape juice [20–23]. In addition, catechins and CA are

absorbed into the blood stream [24–26]. Thus, it is of interest to

Abbreviations used: 4-POBN, α-(4-pyridyl-1-oxide)-N-tert-butylnitrone; CA, caffeic acid ; 13-HPODE, 13-hydroperoxide octadecadienoic acid.1 e-mail : chem1!wakayama-med.ac.jp

(-dopa), 30³2% (dopamine), 49³2% (-adrenaline), 24³2%

(-noradrenaline), and 54³5% (o-dihydroxybenzene) of the

control, respectively. The high performance liquid chroma-

tography–electron spin resonance (HPLC–ESR) and high per-

formance liquid chromatography–electron spin resonance-mass

spectrometries (HPLC–ESR–MS) showed that caffeic acid in-

hibited the formation of octanoic acid radical and pentyl radical

to 42³2% and 52³7% of the control, respectively. On the

other hand, the polyphenols and their related compounds had

few inhibitory effects on the radical formation in the presence of

EDTA. Visible absorbance measurement revealed that all the

polyphenols exhibiting the inhibitory effect chelate ferrous ions.

Above results indicated that the chelation of ferrous ion is

essential to the inhibitory effects of the polyphenols.

Key words: catechin, chlorogenic acid, free radicals, HPLC–

ESR–MS, lipid peroxidation.

examine the influence of polyphenols such as CA, chlorogenic

acid, catechin, on human health in view of their widespread

occurrence in food products and the relatively large quantities

consumed by virtually the entire human population.

There are many reports showing protective effects of the

polyphenols against oxidative stresses. Chlorogenic acid and CA

have been known to be inhibitors of the mutagenicity of bay-

region diol epoxides of polycyclic aromatic hydrocarbons [27], of

retinoic acid 5,6-epoxidation [28], of hydroxyl radical formation

[29], and of lipid peroxidation [30,31]. Chlorogenic acid and CA

also act as scavengers of superoxide radical, hydroxyl radical [32]

and peroxy radical [33]. On the other hand, catechins exert

protective effects against oxidative damage of erythrocyte mem-

brane [34], cardiovascular diseases [35,36], inflammatory [37] and

cancer [38]. Liu and Mori reported that monoamine metabolites,

i.e. norepinephrine and dopamine provide an antioxidant de-

fence in the brain against oxidant-and free radical-induced

damage [39]. Neuromelanin also showed a distinct protective

effect on lipid peroxidation induced by ferrous ions or water-

soluble free-radical initiator, 2,2«azobis(amidinopropane)di-

hydrochloride [40]. These protective effects seem to be mainly

attributed to their antioxidative activities by preventing the

formation of free radicals [41].

However, little is known about the effects of the polyphenols

on the formation of respective lipid-derived free radicals. In this

study, two kinds of lipid-derived free radicals, i.e. pentyl radical

and octanoic acid radical, which form in the reaction of 13-

hydroperoxide octadecadienoic acid (13-HPODE) with ferrous

ions are separated and identified using the high performance

# 2000 Biochemical Society

266 H. Iwahashi

liquid chromatography–electron spin resonance–mass spec-

trometry (HPLC–ESR–MS) [42]. Furthermore, effects of some

polyphenols on the formation of the respective lipid-derived free

radicals are examined.

MATERIALS AND METHODS

Materials

Linoleic acid (9,12-octadecadienoic acid), α-(4-pyridyl-1-oxide)-

N-tert-butylnitrone (4-POBN), -(®)-catechin, and soybean

lipoxygenase (EC 1.13.11.12) Type V were obtained from Sigma

Chemical Co. (St. Louis, MO, U.S.A.). Chlorogenic acid, caffeic

acid (CA), quinic acid, ferulic acid, gallic acid, -()-catechin, 4-

hydroxy-3-methoxybenzoic acid, -dopa, dopamine hydrochlo-

ride, -adrenaline, -noradrenaline, o-dihydroxybenzene, m-di-

hydroxybenzene and p-dihydroxybenzene were purchased from

Tokyo Kasei Kogyo, Ltd. (Tokyo, Japan). Salicylic acid was

from Katayama Chemical Co. (Osaka, Japan). Ferrous am-

monium sulphate was obtained from Kishida Chem. Co. (Osaka,

Japan). Ettylenediaminetetraacetic acid disodium salt (EDTA)

was obtained from Wako Pure Chemistries, Ltd. (Osaka, Japan).

Pentylhydrazine oxalate was synthesized according to the method

of Gever and Hayes [43]. All other chemicals used were of

analytical grade.

Preparation of 13-hydroperoxide octadecadienoic acid (13-HPODE)

The reaction mixture contained, in total volume of 25 ml,

1±5 mg}ml linoleic acid, 440 units}ml soybean lipoxygenase, and

0±2 M boric acid (pH 9±0). Reaction was performed at 25 °Cunder air for 1 h. After 1 h reaction, 0±4 ml of the reaction

mixture was mixed with 3±6 ml of 0±2 M borate buffer (pH 9±0),

and then injected onto an HPLC-UV system. The HPLC-UV

used consisted of a model 7125 Rheodyne injector (Reodyne,

Cotati, CA, U.S.A.), a model Hitachi 655A-11 pump with a

model L-5000 LC controller (Hitachi Ltd., Ibaragi, Japan), a

Water µBondapak C")

semipreparative column (Millipore Co.,

Milford, MA, U.S.A.) (30 mm¬10 mm I.D.), and a model SPD-

M10AVP diode array detector (Shimadzu Co., Kyoto, Japan)

with a model CLASS-LC10 LC workstation (Shimadzu Co.,

Kyoto, Japan). The SPD-M10AVP diode array detector was

operated at 200–350 nm in the HPLC-UV system. Two solvents

were used in the HPLC-UV: A, water ; B, methanol. A com-

bination of isocratic and linear gradient was used for the HPLC-

UV: 0–20 min, 0–90% B (linear gradient) at flow rate 2±0 ml;

20–30 min, 90% B (isocratic) at flow rate 2±0 ml}min. A promi-

nent peak was observed at a retention time of 20±3 min when the

HPLC profile was monitored at 235 nm. The peak fraction was

collected. The methanol contained in the fraction was removed

using a model CC-105 centrifugal concentrator with a model

TU-055 Low Temperature Trap (Tomy Ltd., Yokohama, Japan).

The water solution of the fraction was used as a stock solution

of 13-HPODE [44]. The concentration of 13-HPODE was

determined from its absorbance at 234 nm (ε¯ 25600 M−"[cm−")

[45].

Control reaction mixture of 13-HPODE with ferrous ions

The control reaction mixture of 13-HPODE with ferrous ions

contained 140 µM 13-HPODE, 0±33 mM FeSO%(NH

%)#SO

%,

0±1 M 4-POBN, and 38 mM phosphate buffer (pH 7±4). The

reaction was started by adding FeSO%(NH

%)#SO

%. The reaction

was performed for 2 min at 25 °C.

Figure 1 ESR spectra of the reaction of 13-HPODE with ferrous ions

The reaction and ESR conditions were as described in the Materials and methods section. A,Control reaction mixture ; B, control reaction mixture without ferrous ions ; C, control reaction

mixture without 13-HPODE.

ESR measurements

The ESR spectra were obtained using a model JES-FR30 Free

Radical Monitor (Jeol Ltd., Tokyo, Japan). Aqueous samples

were aspirated into a Teflon tube centred in a microwave cavity.

Operating conditions of the ESR spectrometer were: power,

4 mW; modulation width, 0±1 mT; centre of magnetic field,

337±200 mT; sweep time, 4 min; sweep width, 10 mT; time

constant, 0±3 s. Magnetic fields were calculated by the splitting of

MnO (∆H$–%¯ 8±69 mT).

Visible absorption spectra

Visible absorption spectra were measured using a model UV-

160A ultraviolet-visible spectrophotometer (Shimadzu Co.,

Kyoto, Japan). The spectrophotometer was operated from

400 nm to 800 nm. The measurements were performed at 25 °C.

In the reference cell, 38 mM phosphate buffer (pH 7±4) was

contained. Sample solutions consisted of 38 mM phosphate

buffer (pH 7±4), 1 mM chlorogenic acid (or CA, or quinic acid, or

ferulic acid, or gallic acid, or -()-catechin, or -(®)-catechin,

or 4-hydroxy-3-methoxybenzoic acid, or salicylic acid, or -

dopa, or dopamine, or -adrenaline, or -noradrenaline, or o-

dihydroxybenzene, or m-dihydroxybenzene, or p-dihydroxy-

benzene), and 0±33 mM FeSO%(NH

%)#SO

%.

HPLC–ESR analysis

The HPLC used in the HPLC–ESR consisted of a model 7125

injector (Reodyne, Cotati, CA, U.S.A.) with a 5 ml sample loop,

a model 655A-11 pump with a model L-5000 LC controller

(Hitachi Ltd., Ibaragi, Japan). A Water µ Bondapak C")

semi-

preparative column (30 mm¬10 mm I.D.) (Millipore Co.,

Milford, MA, U.S.A.) was used. The column was kept at

25 °C throughout the analyses. For the HPLC–ESR analyses,

two solvents were used: solvent A, 50 mM ammonium acetate ;

solvent B, 50 mM ammonium acetate}acetonitrile (20:80, v}v).

A combination of isocratic and linear gradient was used:

0–30 min, 100% A to 20% A (linear gradient) at flow rate

2±0 ml}min; 30–40 min, 20% A (isocratic) at flow rate 2±0 ml}min. The eluent was introduced into a model JES-FR30 Free


267Polyphenols inhibit the formation of pentyl and octanoic acid radicals

Figure 2 ESR spectra of the reaction of 13-HPODE with ferrous ions in thepresence of CA

The reaction and ESR conditions were as described in the Materials and methods section.

Amplitude of the ESR spectra D and E was half of the ESR spectra A, B, and C. A, Control

reaction mixture. B, 1 mM CA was added to the control reaction mixture at 0 min. C, 1 mM

CA was added to the control reaction mixture at 1±5 min. D, Control reaction mixture with

1 mM EDTA. E, CA was added to the control reaction mixture with 1 mM EDTA at 0 min.

Radical Monitor (Jeol Ltd., Tokyo, Japan). The ESR spec-

trometer was connected to the HPLC with a Teflon tube, which

passed through the centre of the ESR cavity. The operating

conditions of the ESR spectrometer were: power, 4 mW; modu-

lation width, 0±2 mT; time constant, 1 s. The magnetic field was

fixed at the third ESR peak indicated by arrow (Figure 1)

throughout the experiments.

Synthesis of pentyl radical

Authentic pentyl radical was synthesized through the decompo-

sition of pentylhydrazine. The reaction mixture contained, in

total volume of 1 ml, 0±1 M 4-POBN, 2±5 mg pentylhydrazine

oxalate, 0±2 mM CuCl#, and 45 mM carbonate buffer (pH 10±0).

After nitrogen gas was bubbled through the reaction mixture

without CuCl#

for 5 min, the reaction was started by adding

CuCl#. Reaction was performed for 2 h at 25 °C. 10 µl of the

pentyl radical reaction mixture was mixed with 1±49 ml of 50 mM

phosphate buffer (pH 7±4) and then applied to the HPLC–ESR.

Figure 3 Time course of 4-POBN/13-HPODE radical adduct formation

The reaction and ESR conditions were as described in the Materials and methods section. (D),

Control reaction mixture. (E), 1 mM CA was added to the control reaction mixture at 0 min.

(*), 1 mM CA was added to the control reaction mixture at 1±5 min. (^), The control reaction

mixtures with 1 mM EDTA. (_), 1 mM CA was added to the control reaction mixture with

1 mM EDTA at 0 min.

HPLC–ESR–MS analysis of the peak 1 and peak 2 radicaladducts

The HPLC–ESR–MS consisted of a model 7125 injector

(Reodyne Cotati, CA, U.S.A.), a model L-7100 pump (Hitachi

Ltd., Ibaragi, Japan), a Water µ Bondapak C")

semi-preparative

column (30 mm¬10 mm I.D.) (Millipore Co., Milford, MA,

U.S.A.), a model JES-FR30 Free Radical Monitor (Jeol Ltd.,

Tokyo, Japan), and a model M-1200AP LC–MS system with an

electrospray ionization (ESI) (Hitachi Ltd., Ibaragi, Japan). The

HPLC and ESR conditions in the HPLC–ESR–MS analysis

were same as those in the HPLC–ESR analysis except for the

HPLC mobile phase. For the HPLC–ESR–MS analyses, two

solvents were used: solvent A, 50 mM acetic acid; solvent B,

50 mM acetic acid}acetonitrile (20:80, v}v). A combination of

isocratic and linear gradient was used: 0–30 min, 100% A

to 20% A (linear gradient) at flow rate 2±0 ml}min; 30–40 min,

20% A (isocratic) at flow rate 2±0 ml}min. The operating

conditions of the mass spectrometer were: nebulizer, 180 °C;

aperture 1, 120 °C; N#

controller pressure, 2±0 kgf}cm# ; drift

voltage, 70 V; multiplier voltage, 1800 V; needle voltage, 3000 V;

polarity, positive; resolution, 48.


268 H. Iwahashi

Figure 4 Structures of polyphenols and their related compounds

The reaction mixture contained, in total volume of 1±5 ml,

140 µM 13-HPODE, 0±33 mM FeSO%(NH

%)#SO

%, 0±1 M 4-

POBN, 1 mM EDTA and 38 mM phosphate buffer (pH 7±4).

The reaction was started by adding FeSO%(NH

%)#SO

%. After

2 min reaction at 25 °C, the reaction mixture was applied to the

HPLC–ESR. Peaks from three HPLC–ESR chromatograms were

collected and combined. After the volume of the combined

sample had been reduced to about 1 ml, the HPLC–ESR–MS

analysis was performed. The mass spectra of the peaks 1 and 2

were obtained by introducing the eluent from the ESR detector

into the LC–MS system just before the respective peaks were

eluted. The flow rate was kept at 50 µl}min while the eluent was

introducing into the LC–MS system.

RESULTS

ESR measurements of the reaction mixtures of 13-HPODE withferrous ions

ESR spectra of the control reaction mixture, the control reaction

mixture without 13-HPODE, and the control reaction mixture

without ferrous ions were measured (Figure 1). An ESR spectrum

(aN ¯ 1±58 mT and aHβ¯ 0±26 mT) was observed in the control



Figure 5 Effects of polyphenols and their related compounds on theformation of 13-HPODE-derived radicals

The ESR spectra were observed for the control reaction mixture with 1 mM of chlorogenic acid

(or CA, or quinic acid, or ferulic acid, or gallic acid, or D-()-catechin, or D-(®)-

catechin, or 4-hydroxy-3-methoxybenzoic acid, or salicylic acid, or L-dopa, or dopamine, or L-

adrenaline, or L-noradrenaline, or o-dihydroxybenzene, or m-dihydroxybenzene, or p-dihydroxy-benzene). The reaction was started by adding FeSO4(NH4)2SO4. The reaction was performed for

2 min at 25 °C. Signal intensities were evaluated from the peak height of the third ESR signal

of the 4-POBN/13-HPODE-derived radical adducts. The control value 100% represents the

level of 4-POBN/13-HPODE-derived radical adducts formed in the absence of the compounds.

The respective values are means³SD of five determinations. ESR conditions were as described

in the Materials and methods section.

reaction mixture (Figure 1A). ESR peaks were hardly observed

in the absence of ferrous ions (or 13-HPODE) (Figures 1B and

1C).

Effects of CA on the formation of the 13-HPODE-derived radicals

In order to examine the effects of CA on the overall formation of

13-HPODE-derived radicals, ESR spectrum of the control re-

action mixture with 1±0 mM CA was measured (Figure 2B). On

addition of CA to the control reaction mixture, ESR peak height

decreased to 54³2% of the control. To check whether or not

CA inhibits the formation of 13-HPODE-derived radicals or

degrades the radical adducts of 4-POBN with 13-HPODE-

derived radicals, CA was added into the control reaction mixture

at 1±5 min after the reaction was started. Addition of CA at

1±5 min resulted in no effects on the ESR peak height, indicating

that CA inhibits 13-HPODE-derived radicals formation itself

(Figure 2C). To know whether or not chelation of Fe#+ ions is

essential to the inhibitory effects, EDTA was added to the

control reaction mixture (Figure 2D). On addition of EDTA to

the control reaction mixture, the ESR peak height increased

to 270³30% of the control, indicating that EDTA}ferrous ion

complex is more reactive than phosphate ion}ferrous ion complex

in the reaction mixture. Addition of CA into the control reaction

mixture with EDTA resulted in little effect on the ESR peak

height (96³6%) (Figure 2E). These results indicated that CA

inhibits the formation of 13-HPODE-derived radicals through

the chelation of iron ions.

Figure 6 HPLC–ESR analysis of the reaction of 13-HPODE with ferrousions

The reaction and HPLC–ESR conditions were as described in the Materials and methods

section. A, 1±5 ml of control reaction mixture ; B, 1±5 ml of control reaction mixture with 1 mM

CA ; C, 1±5 ml of control reaction mixture with 1 mM EDTA ; D, 1±5 ml of control reaction

mixture with 1 mM EDTA and 1 mM CA.

Time course experiments

Time course experiments of the ESR peak height were performed

(Figure 3). For the control reaction mixture, the reaction reached

plateau at 2 min after the reaction was started. When CA was

added into the control reaction mixture at 0 min, smaller ESR

peaks were observed compared with the control during time

course experiment. The reaction also reached a plateau in 2 min.

The ESR peak heights of the 4-POBN}13-HPODE-derived

radical adducts remained unchanged for the control reaction

mixture with CA during a further 28 min incubation, indicating

that the ESR spin adducts are stable in the reaction mixture.

When CA was added into the control reaction mixture at

1±5 min, the ESR peak height was almost the same as the control.

The ESR peak height also remained unchanged during a further

28±5 min incubation. CA apparently does not convert the 4-

POBN}13-HPODE-derived radical adducts to ESR silent

species. The above results indicated that CA inhibits 13-HPODE-

derived radical formation itself.

Time course experiment was performed for the control reaction

mixture with EDTA in the presence of CA (or in the absence of

CA). The control reaction mixture with EDTA reached a plateau

in 7 min. On the other hand, when CA was added into the

control reaction mixture with EDTA, the ESR peak height

continued to increase during a 28 min incubation.

Effects of some polyphenols and their related compounds on theformation of the 13-HPODE-derived radicals

Effects of some polyphenols and their related compounds (Figure

4) on the overall formation of the 4-POBN}13-HPODE-derived


270 H. Iwahashi

Figure 7 The HPLC–ESR analysis of control reaction mixture and thereaction mixture of authentic 4-POBN/pentyl radical adduct

The reaction and HPLC–ESR conditions were as described in the Materials and methods

section. A, 1±5 ml of control reaction mixture ; B, 10 µl of reaction mixture of authentic 4-

POBN/pentyl radical adduct was mixed with 1±49 ml of 50 mM phosphate buffer (pH 7±4) and

applied to the HPLC–ESR ; C, 1±49 ml of control reaction mixture was mixed with 10 µl of

reaction mixture of authentic 4-POBN/pentyl radical adduct and applied to the HPLC–ESR.

Figure 8 HPLC–ESR–MS analysis of the peak 1 and peak 2 fractions

The reaction and HPLC–ESR–MS conditions are as described in the Materials and methods

section. A, peak 1 ; B, peak 2.

radical adducts were examined (Figure 5). The ESR peak heights

of the 4-POBN}13-HPODE-derived radical adducts decreased

to 46³4% (chlorogenic acid), 49³2% (gallic acid), 55³1% [-

Figure 9 Effect of oxygen concentration on the formation of peak 1 andpeak 2 radical adducts

The HPLC–ESR conditions are as described in the Materials and methods section. After

nitrogen gas was bubbled into the control reaction mixture without FeSO4(NH4)2SO4 for 5 min,

the reaction was started by adding FeSO4(NH4)2SO4. The reaction was performed for 2 min at

25 °C. 1±5 ml of the reaction mixture was applied to the HPLC–ESR analysis. A, control

reaction mixture under air ; B, control reaction mixture under nitrogen gas.

()-catechin], 60³3% [-(®)-catechin], 42³1% (-dopa),

30³2% (dopamine), 49³2% (-adrenaline), 24³2% (-nor-

adrenaline), and 54³5% (o-dihydroxybenzene) of the control,

respectively. On the other hand, quinic acid (99³4%), ferulic

acid (104³5%), 4-hydroxy-3-methoxybenzoic acid (93³4%),

salicylic acid (91³5%), m-dihydroxybenzene (107³5%), and p-

dihydroxybenzene (94³4%) showed no effects on the overall

formation of the 4-POBN}13-HPODE-derived radical adducts.

Visible absorption spectra of the mixtures of ferrous ions withsome polyphenols and related compounds

Visible absorption spectra were measured for the mixtures of

Fe#+ ions with chlorogenic acid (or other polyphenols and related

compounds). Of the polyphenols and their related compounds,

chlorogenic acid, CA, gallic acid, -()-catechin, -(®)-

catechin, -dopa, dopamine, -adrenaline, -noradrenaline, and

o-dihydroxybenzene showed characteristic absorbance bands

around 550 nm. The absorbance bands were not observed for the

mixtures without ferrous ions (or the polyphenols), indicating

the formation of polyphenol chelates of Fe#+ ions. The λmaxS

(nm) and absorbances of the polyphenol chelates of Fe#+ ions are

as follows: chlorogenic acid [488 nm (1±37) and 640 nm (1±12)],

CA [591 nm (0±859)], gallic acid [552 nm (0±617)], -()-

catechin [559 nm (0±584)], -(®)-catechin [562 nm (0±630)], -

dopa [559 nm (0±552)], dopamine [546 nm (0±496)], -adrenaline

[534 nm (0±768)], -noradrenaline [532 nm (0±773)], and o-

dihydroxybenzene [562 nm (0±292)].

The HPLC–ESR analysis of the control reaction mixture, thecontrol reaction mixture with CA, the control reaction mixturewith EDTA, and the control reaction mixture with EDTA and CA

In order to know the effect of CA on the formation of respective

13-HPODE-derived radicals, the HPLC–ESR analyses were

performed for the control reaction mixture, the control reaction

mixture with CA, the control reaction mixture with EDTA, and

the control reaction mixture with EDTA and CA. On the

HPLC–ESR elution profile of the control reaction mixture, two



Scheme 1 Proposed mechanism for the formation of pentyl radical and octanoic acid radical in the reaction of 13-HPODE with ferrous ions

prominent peaks were observed at the retention times of

22±1³0±3 min (peak 1) and 31±3³0±3 min (peak 2), respectively

(Figure 6A). When CA was added to the control reaction

mixture, respective peak heights decreased to 42³2% (peak 1)

and 52³7% (peak 2) of the control (Figure 6B), indicating that

CA inhibits the formation of the peak 1 and peak 2 compounds,

respectively. On the other hand, on addition of EDTA to the

control reaction mixture, respective peak heights increased to

150³20% (peak 1) and 170³30% (peak 2) of the control

(Figure 6C). In the presence of EDTA, addition of CA to the

control reaction mixture resulted in little effect on the formation

of the peak 1 (90³8%) compound and peak 2 compound

(89³8%) (Figure 6D).

In order to identify the peak 1 and peak 2 compounds, 10 µl

of authentic pentyl radical reaction mixture was mixed with

1±49 ml of 50 mM phosphate buffer (pH 7±4) and then the

HPLC–ESR analysis was performed for the solution (Figure

7B). The 4-POBN}pentyl radical adduct was eluted at the

retention time of 31±4 min. The retention time is identical with

peak 2 compound. When 1±5 ml of the control reaction mixture

was mixed with 10 µl of authentic pentyl radical reaction

mixture and then applied to the HPLC–ESR, peak height of the

peak 2 compound increased (Figure 7C). Thus, peak 2 compound

was shown to be 4-POBN}pentyl radical adduct.

The HPLC–ESR–MS analysis of the control reaction mixture withEDTA

To identify the radical species formed in the control reaction

mixture with EDTA, the HPLC–ESR–MS experiments were


272 H. Iwahashi

performed [48]. Mass spectra of peak 1 and peak 2 fractions are

shown in Figure 8.

The HPLC–ESR–MS analysis of peak 1 compound gave ions

at m}z 251, m}z 338 and m}z 675, respectively. The ion m}z 338

corresponds to the protonated molecular ion of 4-POBN}octanoic acid radical adduct, (MH)+. A fragment ion at

m}z 251 corresponds to the loss of [(CH$)$C(O)N] from the

protonated molecular ion. The ion m}z 675 is the protonated

dimer ion, (2MH)+.

The HPLC–ESR–MS analysis of peak 2 compound gave ions

at m}z 179, m}z 266 and m}z 531, respectively. The ion m}z 266

corresponds to the protonated molecular ion of 4-POBN}pentyl

radical adduct, (MH)+. A fragment ion at m}z 179 corresponds

to the loss of [(CH$)$C(O)N] from the protonated molecular ion.

The ion m}z 531 is the protonated dimer ion, (2MH)+.

Effects of oxygen concentration on the formation of peak 1 andpeak 2 radical adducts

Effects of oxygen concentration on the formation of peak 1 and

peak 2 radical adducts were examined using the HPLC–ESR

(Figure 9). Peak 1 radical adduct was predominant under air. On

the other hand, peak 2 radical adduct was predominant under

nitrogen gas.

DISCUSSION

In this study, 4-POBN}octanoic acid radical adduct and 4-

POBN}pentyl radical adduct were detected and identified in

the reaction mixture of 13-HPODE with ferrous ions using the

HPLC–ESR–MS. Possible reaction paths for the formation of

these radicals are shown in Scheme 1. Our previous studies have

shown the formation of octanoic acid radical and pentyl radical

in the reaction mixture of linoleic acid with soybean lipoxygenase

[12,14]. Product analysis and spin-trapping studies provided

evidence for the formation of 13-alkoxylinoleic acid radical

through the reaction of 13-HPODE with ferrous ions [7–9]. The β

scissionof13-alkoxylinoleicacid radical results in the formationof

pentyl radical [11–14]. The pentyl radical could be a precursor

of pentane. Garssen et al. [46] have reported the formation of

pentane and 13-oxo-9,11-tridecadienoic acid in the reaction

mixture of soybean lipoxygenase with linoleic acid. On the other

hand, 13-alkoxylinoleic acid radical possibly rearranges intra-

molecularly by addition to a double bond to cause the formation

of 12,13-epoxylinoleic acid radical. Indeed, 12,13-epoxylinoleic

acid radical was detected and identified in the reaction mixture of

soybean lipoxygenase with linoleic acid in borate buffer (pH 9±0)

[14]. In this study, however, 4-POBN}12,13-epoxylinoleic acid

radical adduct was not detected by the HPLC–ESR–MS. It may

be due to the different reaction conditions. Phosphate buffer

(pH 7±4) was employed in this study. On the other hand, 4-

POBN}12,13-epoxylinoleic acid radical adduct was detected in

borate buffer (pH 9±0) [14]. The reaction of 12,13-epoxylinoleic

acid radical with oxygen molecule results in the formation of 12,

13-epoxy-9-hydroperoxylinoleic acid radical. Gardner et al. [47]

have reported that 12,13-epoxy-9-hydroperoxylinoleic acid forms

in the reaction mixture of 13-HPODE with cysteine-FeCl$.

Octanoic acid radical (peak 1) possibly forms through β scission

of 12,13-epoxy-9-alkoxylinoleic acid radical. The reaction be-

tween 13-alkoxylinoleic acid radical and 12,13-epoxylinoleic acid

radical seems to be reversible [48] (Scheme 1) because the

HPLC–ESR–MS peak height of 4-POBN}pentyl radical adduct

increased under nitrogen gas (Figure 9).

Polyphenols possibly inhibit the following two steps, i.e. step

1, the reaction between 13-HPODE and 13-alkoxylinoleic acid

radical, and step 2, the reaction between 12,13-epoxy-9-hydro-

peroxylinoleic acid and 12,13-epoxy-9-alkoxylinoleic acid radical

because ferrous ions participate in the two reactions. Formation

of octanoic acid radical is inhibited at both steps in the reaction

paths. On the other hand, formation of pentyl radical is inhibited

only at step 1. The HPLC–ESR peak heights decreased to

42³2% (octanoic acid radical) and 52³7% (pentyl radical) of

the control. Formation of octanoic acid radical was more

effectively inhibited. The measurement is consistent with the

above reaction paths. The HPLC–ESR–MS analysis allowed us

to examine effects of the polyphenols on the respective radical

formations.

This study was financed by the Special Co-ordination Fund for Promoting Scienceand Technology of the Science and Technology Agency of the Japanese Government.

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some polyphenols inhibit the formation of pentyl radical and octanoic acid radical

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